Drug Binding

At the molecular level, a number of factors contribute to the interaction between drug and receptor and control the strength, duration, and type of the drug-receptor interaction. Collectively these factors dictate the strength with which the drug forms a complex with its receptor, also known as the affinity:

Size and shape of the drug molecule

Types, number, and spatial arrangement of drug binding sites (stereochemistry)

Intermolecular forces between drug and binding sites

Van der Waals forces = weak bonds and transient reversible effects

Hydrogen bonds = intermediate bonds and transient reversible effects

Covalent bonds = strong bonds and long-lasting or irreversible effects

It is important to recognize that, in most cases, binding of drug to target molecules involves weaker bonds. Accordingly, the drug-receptor complex is not static, but rather there is continuous association and dissociation of the drug with the receptor as long as drug is present. A measure of the relative ease with which the association and dissociation reactions occur is the equilibrium dissociation constant (K_D). Each drug-receptor combination will have a characteristic K_D value. Drugs with high affinity for a given receptor display a small value for K_D, and vice versa. In Figure 1-1, A and B, Drug A has a higher affinity for the receptor than Drug B. K_D also represents the concentration of drug needed to bind 50% of the total receptor population. These concepts are important in the study of basic pharmacologic data regarding different compounds with affinity for the same receptor. In general, drugs with lower K_D values will require lower concentrations to achieve sufficient receptor occupancy to exert an effect.

Selectivity of Drug Responses

Another important and desirable facet of pharmacologic responses is selectivity of drug action, determined by drug molecules exhibiting preferential affinity for receptors, as follows:

The cell will respond only to the spectrum of drugs that exhibit affinity for the receptors expressed by the cell.

The greater the extent to which a drug molecule exhibits high affinity for only one receptor, the more selective will be the drug’s actions, with lower potential for side effects.

The higher the affinity and efficacy of a given drug, the smaller the amount of drug necessary to activate a critical mass of drug receptors to effect a tissue response, and the lower the potential for nonselective actions.

It is important to note that selectivity of drug action is a key concept. Few drugs are entirely specific for one receptor. Rather, drugs exhibit selectivity toward different receptors based on their relative affinities. Thus, selectivity is also relative. As the concentration of a drug increases, the drug will combine with receptors for which it has lower affinity and may generate off-target effects.

Clinical Connection: β-Adrenergic receptor antagonists are effective drugs for a number of cardiovascular disorders. Some β-adrenergic receptor antagonists are selective for β₁-adrenergic receptors to limit the potential for bronchoconstriction caused by blocking β₂-adrenergic receptors. However, even β₁-selective antagonists must be used cautiously in asthmatic patients, particularly at higher doses, to avoid further impairment of airway function in these patients.

Tissue Distribution of Receptors

Only those tissues possessing receptors will respond to the drug.

The more restricted the distribution of drug receptor, the more selective will be the effects of drugs that interact with that receptor.

Clinical Connection: Knowledge of receptor subtypes and their regional distribution can assist in drug selection. A useful example is the use of α-adrenergic receptor antagonists for the treatment of urinary retention secondary to prostatic hypertrophy. Nonselective α-adrenergic antagonists are not routinely used to treat urinary retention in men with prostatic hypertrophy, because although they block α receptors in the prostate and improve urine flow, they also block α receptors in blood vessels and cause hypotension. The prostate expresses primarily α_1B-adrenergic receptors, whereas blood vessels express other subtypes. Consequently, drugs such as tamsulosin that are selective for the α_1B subtype expressed primarily in the prostate are much more useful in the treatment of prostatic hypertrophy.

Activation of the Molecular Target

The relationship between the drug-receptor binding event and the ultimate biologic effect is complex. Quite often in experimental settings, the K_D (concentration causing 50% receptor occupancy) does not correspond to a 50% maximal response from the test tissue or organism. In fact, in many cases half-maximal tissue responses are obtained at drug concentrations below the K_D, suggesting that amplification of drug response occurs. Amplification of drug responses is discussed in a later section. This observation suggests that other factors, in addition to affinity and receptor occupancy, determine the strength of response. Accordingly, an additional modifier termed intrinsic activity was proposed. Intrinsic activity indicates the ability of receptor-bound drug to activate the receptor and initiate downstream events, leading to an effect. Drugs are categorized based on their intrinsic activity at a given receptor:

Agonists (sometimes called full agonists) produce maximum activation of the receptor and elicit a maximum response from the tissue. They are assigned an intrinsic activity of 1.

Antagonists bind but produce no activation of the receptor and therefore block responses from the tissue. They are assigned an intrinsic activity of 0.

Partial agonists exhibit intrinsic activity between 0 and 1. Partial agonists produce weaker activation of the receptor than full agonists or the endogenous ligand. Partial agonists produce only partial activation of the receptor and its downstream signaling events. The clinical effect of a partial agonist will depend on its intrinsic activity and the concentration of the endogenous ligand. If concentrations of the endogenous ligand are really low, then a partial agonist will increase receptor activation, functioning as a weak agonist. In contrast, if concentrations of endogenous ligand are high, the partial agonist will compete for receptors and bind to a certain proportion of receptors previously bound by endogenous ligand. Because the partial agonist produces weaker activation of the receptor than endogenous ligand, the net effect will be less cumulative receptor activation. This will produce inhibition of the response mediated by the endogenous ligand, and the partial agonist will act as a weak antagonist.

Inverse agonists inhibit rather than activate the receptor. This phenomenon is evident with receptors that exhibit baseline (ongoing or constitutive) activity in the absence of agonist binding. In these cases, binding of the inverse agonist reduces the baseline activity of the receptor, which in turn elicits an effect opposite that of binding of the agonist. Inverse agonists and antagonists will elicit similar effects because both types of drugs will reverse the effects of endogenous ligands. Many clinically used antagonists may in fact be inverse agonists. Inverse agonists may assume particular clinical importance in disease states in which constitutive activity of receptors plays an important role. Increasing evidence suggests that a number of diseases are a result of gain of function mutations at the receptor that result in constitutive activity of the receptor in the absence of agonist.

Clinical Connection: Drugs that act as inverse agonists may have important clinical applications for diseases in which receptors are activated in the absence of endogenous agonist. One example is in cancer chemotherapy. In a number of human cancers, mutations of the epidermal growth factor receptor cause the receptor to be active in the absence of epidermal growth factor. In this setting, a traditional antagonist would be of no benefit. However, drugs that act as inverse agonists at the epidermal growth factor would suppress receptor activation and reduce the growth signaling via this pathway. Epidermal growth factor inverse agonists are being studied as cancer chemotherapy drugs.

Thus, the ultimate action of a drug will depend on both its affinity and its intrinsic activity. It is important to remember that affinity and intrinsic activity are distinct properties. A weak partial agonist, which by definition activates a receptor only minimally, may have very high affinity for a receptor. In this case the drug will be able to effectively compete for the receptor and will usually out-compete the endogenous agonist for receptor occupancy and inhibit the endogenous response.

Graded Dose-Response Curves

Measure an effect that is continuous such that, in theory, any value is possible in a given range (0% through 100%).

Have a sigmoidal shape similar to the drug receptor occupancy curves shown in Figure 1-2, because the biologic response to a drug is determined by the interaction of a drug with a receptor or molecular target.

Exhibit a dose beyond which no further response is achieved (maximal effect; E_max). E_max is a measure of the pharmacologic efficacy of the drug.

Show the dose that produces 50% of the E_max (ED₅₀).

ED₅₀ is an index of the potency of the drug.

Agonists with higher potency will have lower ED₅₀ values.

Figure 1-2 A, Graded dose-response curve. B, Quantal dose-response curve.

The ED₅₀ and E_max are useful parameters to assess drugs. In Figure 1-2, A, Drug A is more potent than Drug B or Drug C, whereas Drugs B and C have equal potency. Potency is sometimes used incorrectly as a measure of therapeutic effectiveness. In fact, in most cases potency is secondary to E_max in drug selection. However, in situations in which the absorption of drug is very poor, such that only small quantities of the drug reach the target, potency can be a critical consideration. Drugs with higher E_max values have higher pharmacologic efficacy.

In Figure 1-2, A, Drug B has the greatest efficacy, followed by Drug C, whereas Drug A, despite being the most potent, has the least efficacy. Drug C is equipotent with Drug B but has less efficacy. Thus, potency and efficacy can vary independently. It is important not to confuse the pharmacologic usage of efficacy with the more general usage. Pharmacologic efficacy is a measure of the strength of effect produced by the maximum dose of drug. By definition, antagonists do not activate their receptors after binding and therefore have an intrinsic activity and efficacy of 0. Nevertheless, an antagonist may be very clinically “efficacious” or beneficial because it blocks activation of the receptor by endogenous agonist.

These variables can be useful in determining how much of a drug to administer. For example, knowledge of the ED₅₀ concentration for blood pressure lowering can be used to determine the dose of antihypertensive agent to administer to achieve a certain magnitude of blood pressure reduction. However, the astute clinician recognizes that ED₅₀ values are derived from the average of a great many patients and thus should be used only as initial guidelines. Because of interindividual variability, each patient may respond in ways that differ from the average. The second type of dose-response curves, quantal dose-response curves, provide an estimate of this variability.

Quantal Dose-Response Curves

Quantal dose-response curves do the following:

Quantify responses for variables that are all or none (e.g., seizure or no seizure).

Describe the relationship between drug dosage and the frequency with which a biologic effect occurs. For example, in individuals administered an anticonvulsant medication, the percentage of individuals not experiencing a convulsive episode at any given dose is plotted in cumulative fashion (Figure 1-2, B).

Represent a cumulative frequency distribution for a given response.

Provide an ED₅₀ value that reflects the dose of drug that produces a response in 50% of the population (also called the median effective dose).

Provide an E_max value that is the dose at which all of the patients respond to the drug.

Provide an estimate of the variability in response of the patient population to the drug. A steep slope indicates that all the patients respond in a narrow range of doses, whereas a shallow slope indicates considerable variability in the ability of the drug to elicit a response in the patient population.

Can be constructed for a graded or continuous response by choosing a target magnitude of response (e.g., blood pressure reduction of 20 mm Hg) and plotting the proportion of patients that achieves this magnitude of response at each increase in dosage. This type of information can be useful in determining a starting dosage to achieve a given level of effect

Can be plotted for therapeutic, toxic, and lethal effects to obtain:

• TD₅₀ (median toxic dose), is the dose that produces toxic effects in 50% of the patient population.

• LD₅₀ (median lethal dose), the dose at which 50% of patients die

• Comparison of these parameters can provide an estimate of the relative safety of treatment.

• Therapeutic index (TI) or therapeutic ratio is the ratio of the LD₅₀ and ED₅₀. Large values of TI are desirable because they indicate that the doses that produce death are much greater than those that produce a therapeutic effect.

• Therapeutic window is a loosely defined term that generally refers to the range of doses that produce therapeutic effects with minimal toxic effects. It can be viewed as the lack of overlap between the quantal dose-response curves for therapeutic and toxic or lethal effects. There are several indices of the degree of this overlap.

• Certain safety factor: The certain safety factor is the dose of drug that produces a lethal effect in 1% of the population (LD₁) divided by the dose that produces a therapeutic effect in 99% of the population (ED₉₉) (LD₁/ED₉₉).

• Protective index: The protective index is calculated as the dose for an undesirable effect in 50% of patients (TD₅₀) divided by the ED₅₀ for the desired effect (TD₅₀/ED₅₀).

• For both the certain safety factor and the protective index, large values signify that there is little overlap between the therapeutic and toxic or lethal effects of the drug, and thus there is a relatively large margin of safety for its use.

Chemical (Physical) Antagonists

Chemical antagonists do not act at the receptor level, but rather there is a chemical or physical interaction between the drug and the endogenous target substance. A routinely used example is antacid used in the treatment of heartburn. Most antacids are basic magnesium, aluminum, or calcium salts that react with and neutralize gastric acid.

Physiologic (Functional) Antagonists

Physiologic antagonists represent another type of antagonism in which the antagonist does not interact directly with the actions of the agonist at its molecular target.

The agonist and antagonist each act on different molecular targets, but the responses elicited by these interactions are diametrically opposed and negate each other.

Epinephrine and histamine are good examples of physiologic antagonists. Histamine is a vasodilator and bronchoconstrictor. Histamine release in anaphylactic reactions can cause hypotension and respiratory compromise. Epinephrine supports blood pressure and causes bronchodilation but does not act through the histamine receptor. Thus, epinephrine is given as acute treatment for anaphylactic reactions in part because it is a physiologic antagonist to histamine.

Pharmacokinetic Antagonists

One drug attenuates the action of another drug by decreasing its concentration at the site of action.

This may occur through changes in absorption, distribution, metabolism, or excretion.

An example is activated charcoal used in acute treatment of poisonings. Ingestion of activated charcoal binds drug in the intestine and reduces or prevents its absorption.

Pharmacologic Antagonists

The majority of antagonists used as drug therapy are pharmacologic antagonists that act by directly interfering with an agonist’s ability to activate its molecular target. The antagonist prevents agonist binding or agonist activation of the receptor and inhibits the biologic effects generated by the agonist. The interaction between antagonist and agonist can take several forms, including competitive reversible, competitive irreversible, and noncompetitive antagonism.

Competitive reversible antagonism (also called competitive surmountable antagonism)

The antagonist competes directly for the target receptor with the agonist molecule. These interactions will follow the law of mass action and the same general principles described earlier, with the added facet that now two drugs are competing for receptor occupancy.

Because there is a fixed number of receptors (at least in the short term), each antagonist-receptor complex formed removes receptor molecules from the mass action equation and reduces the likelihood of formation of an agonist-receptor complex.

Owing to the constant competition for receptors, the concentrations and affinities for both the agonist and antagonist drugs will determine the overall balance of the mass action equation.

As the concentration of antagonist increases, the number of antagonist-receptor complexes increases and the number of agonist-receptor complexes decreases. Therefore the agonist effect decreases. Figure 1-3, A shows a graded dose-response curve for increasing concentrations of antagonist in the presence of a fixed concentration of agonist. The concentration of antagonist that reduces the agonist response to 50% of maximum is the IC₅₀, one index for quantifying antagonist effectiveness. Note that IC₅₀ values vary with agonist starting concentration.

The greater the affinity of the antagonist, the greater the number of antagonist-receptor complexes formed at any given concentration of antagonist.

The agonist-antagonist relationship can also be depicted on agonist dose-response curves. Figure 1-3, B illustrates graded agonist dose-response curves in the absence (control) and presence of increasing doses of antagonist.

Addition of antagonist causes a reduction of agonist-driven response at any concentration of the agonist. It is important to note, however, that as the agonist concentration is increased, response increases because of greater competition for the receptors by the agonist. Ultimately, an agonist concentration will be reached, at which all of the receptors needed to elicit a maximal response are occupied by agonist and a maximal effect will be elicited.

There is a rightward displacement of the agonist dose-response curve in the presence of a competitive reversible antagonist, but E_max is not affected.

Increasing the concentration of antagonist produces a greater rightward displacement of the agonist dose-response curve, and the ED₅₀ value for the agonist increases progressively. However, a maximal effect can always be reached by increasing the dose of agonist. The rightward parallel displacement of the agonist dose-response curves but with preserved E_max is characteristic of competitive reversible antagonism (see Figure 1-3).

The magnitude of the rightward displacement of the agonist dose-response curve with increasing antagonist doses is an index of the affinity of the antagonist for the receptor and can be quantified by calculating a dose ratio, calculated as the ED₅₀ in the presence of antagonist divided by the ED₅₀ in the absence of antagonist. The dose ratio is used to calculate a pA₂ value, an index of the affinity of the antagonist.

The pA₂ scale is an index of antagonist affinity for the receptor. The pA₂ is the negative logarithm of the dose of antagonist that necessitates a doubling of agonist dose—in other words, a dose ratio of 2.

A lower pA₂ signifies greater antagonist affinity with greater effects at lower doses.

Competitive irreversible antagonism (also called competitive insurmountable antagonism) (in older literature these are also labeled as noncompetitive antagonists)

The antagonist competes directly with the agonist for receptor binding as described previously. However, the binding forces between the antagonist and receptor are so strong that the antagonist-receptor complex is virtually irreversible.

The net effect is that the total number of receptors available to the agonist is permanently reduced (at least until new receptor is synthesized).

An important characteristic of this type of antagonism that distinguishes it from competitive reversible antagonism is that E_max is reduced (Figure 1-4).

A second distinguishing feature of this type of antagonist is that its effects will be constant irrespective of endogenous agonist levels. Thus fluctuations in the release of transmitter or hormone do not affect the response to this type of antagonist. This type of antagonist is useful clinically in situations in which endogenous agonist levels are unpredictable.

Noncompetitive antagonism (also called allotropic or allosteric antagonism)

Noncompetitive antagonists do not directly compete with the agonist for binding at the same binding site but nevertheless impair the ability of an agonist to bind to or activate the receptor, and thus they prevent a response.

An important characteristic of this type of antagonism is that both antagonist and agonist can be bound to the molecular target at the same time.

Because agonists and antagonists bind at distinct sites, increasing concentrations of agonist do not out-compete or reverse the inhibitory action of the antagonist.

Noncompetitive antagonism can occur through a number of mechanisms:

• Reduction in affinity of agonist binding site for the agonist

• Blockade of agonist-induced change in receptor

• Blockade of coupling of receptor to coupling or signaling mechanisms

Noncompetitive antagonists will exert functional effects similar to those of competitive irreversible antagonists in that both types of antagonist will decrease the E_max or efficacy of the agonist.

Because the agonist and antagonist act at different sites on the molecular target, increasing concentrations of agonist cannot overcome the inhibitory action of the antagonist. Thus, the effect of a noncompetitive antagonist will be independent of fluctuations in levels of the endogenous agonist, much like that of a competitive irreversible antagonist.

A clinically relevant difference may be in the duration of action. Most allosteric antagonists combine reversibly with their binding site. Accordingly, discontinuation of the drug will result in a decrease in antagonist binding and effect. In contrast, as mentioned earlier, one of the features of competitive irreversible antagonists is their prolonged duration of action.

Clinical Connection: Drugs that bind covalently to their molecular targets have the benefit of extended duration of action. Knowledge of this characteristic can influence clinical application of the drug. Acetylsalicylic acid (aspirin) irreversibly acetylates and inhibits the cyclooxygenase enzyme present in platelets and thereby inhibits platelet aggregation that contributes to coronary artery occlusion and myocardial infarcts. Although aspirin is cleared relatively quickly from the body, when used for its antiplatelet activity in patients with coronary artery disease, its administration is required only once a day because aspirin does not dissociate from its molecular target when plasma concentrations decrease.

Figure 1-3 A, Competitive reversible antagonism. B, Effect of competitive antagonist on agonist dose-response curves; dose ratio.

Figure 1-4 Competitive irreversible antagonism.

Receptors

In the strictest pharmacologic sense, receptors are molecular entities that evolved specifically to bind certain substances, with the purpose of cellular communication (e.g., cardiac β-adrenergic receptors). The concept of the receptor was introduced at the end of the nineteenth century (Langley, 1878) and beginning of the twentieth century (Ehrlich, 1909). This in turn spurred considerable and ongoing research into the nature of the interaction between drugs and receptors.

General Sites

General sites may not have specifically evolved as communication mechanisms and thus may or may not adhere to all pharmacodynamic principles discussed earlier (e.g., intrinsic activity). Nevertheless, in a general sense these can act as receptors for drug action. Examples of general sites mediating drug action include the following:

Components in key signaling or metabolic pathways

Ion channels or transporters found in the cell membrane

Intracellular or extracellular enzymes

Structural components

Unidentified Targets

Finally, the molecular targets for some drugs have not been completely elucidated yet. An example would be the target for inhaled general anesthetics.

Extracellular Transduction Mechanisms

A number of dugs act outside of the cell to affect cellular function. Generally, these types of drugs act via the following:

Extracellular enzymes. Drugs acting via this mechanism alter the activity of extracellular enzymes involved in the synthesis or degradation of endogenous signaling molecules. These drugs affect the levels of endogenous compounds that then alter cellular function by acting on their receptors. Examples of clinical utility for this mechanism include:

Angiotensin-converting enzyme (ACE) inhibitors, which prevent the formation of angiotensin II and are used in the treatment of hypertension

Inhibition of acetylcholinesterase, which results in increased levels of acetylcholine for the treatment of:

• Neuromuscular disorders

• Glaucoma

Clinical Connection: Myasthenia gravis is an autoimmune disorder that targets the acetylcholine receptor. Loss of these receptors at the neuromuscular junction leads to muscle weakness. Edrophonium, a drug that inhibits the extracellular enzyme (cholinesterase) that degrades acetylcholine, can be used to confirm the diagnosis of myasthenia gravis. Injection of edrophonium produces a short-lived increase in acetylcholine at the neuromuscular junction and an improvement in muscle strength in patients with myasthenia gravis.

Direct interaction with endogenous molecules to affect their ability to reach their sites of action. The clearest example of this type of mechanism is the use of monoclonal antibodies to directly target endogenous signaling molecules. An example of this application is the use of a monoclonal antibody to the cytokine tumor necrosis factor α (TNF-α) (adalimumab) in the treatment of certain autoimmune disorders. Administration of adalimumab binds TNF-α and prevents the cytokine from reaching and activating its receptor.

Transmembrane Transduction Mechanisms

In many cases, the drug or endogenous ligand is a hydrophilic substance that cannot easily cross the plasma membrane of the cell and binds to receptors or other targets embedded in the plasma membrane. Accordingly, mechanisms are needed to transduce or relay the drug signal across the plasma membrane. It is possible to cluster these coupling and transduction mechanisms into several general groups (sometimes called superfamilies).

G protein–coupled receptors (GPCRs). GPCRs, also called seven transmembrane pass receptors, are a large class of receptors that mediate the majority of endogenous transmitter and hormone driven responses (Figure 1-5).

G proteins are trimeric macromolecules that consist of α, β, and γ subunits.

Ligand activation of the receptor causes the GPCR to interact with G proteins.

• G_α is activated by binding of guanosine-5’-triphosphate (GTP) and dissociation of the G_α-GTP complex from the receptor and from its companion βγ subunits.

• Activated G_α-GTP complex then activates downstream effector systems (e.g., adenylate cyclase) to initiate a cascade of cellular events (e.g., cyclic adenosine monophosphate [cAMP] production) that lead to activation of the effector system and the biologic response.

• G_α subunit has intrinsic GTPase activity and eventually hydrolyzes GTP to guanosine diphosphate (GDP), which represents a termination signal for this receptor transduction mechanism.

• G proteins exist in as many as 23 isoforms coupled to different signaling paths. The type(s) of G protein coupled to the receptor determines the response to receptor activation.

• Individual cells may contain receptors coupled to many G proteins.

• The ultimate biologic response to activation of a receptor will be the integrated action of G protein action.

• Approximately 30% of all clinically used drugs interact with GPCR.

Receptor-coupled enzymes. Receptor-coupled enzymes bypass the G protein coupling mechanism and link directly to cellular communication cascades. The receptor is directly coupled in some way to kinase enzymatic activity within the cell. Ligand binding stimulates the kinase enzymatic activity, which then initiates and amplifies intracellular signals and feedback responses by changing the phosphorylation status of cellular proteins. As shown in Figure 1-6, these mechanisms can be grouped into four general types that include receptors:

With integral tyrosine kinase (TK) activity

That recruit TK to the receptor and activate the enzyme

Coupled to serine threonine kinases

With guanylate cyclase activity that generate a second messenger, cyclic guanosine monophosphate (cGMP)

Figure 1-5 G protein–coupled receptors.

Figure 1-6 Receptor-coupled enzymes.

The receptor-coupled enzymes phosphorylate intracellular proteins at tyrosine (Tyr), serine (Ser), or threonine (Thr) residues to change protein function. Alternatively, cGMP generated by guanylate cyclase activates downstream enzymes (effector in Figure 1-6) that change the phosphorylation status of proteins to alter their function. Receptors with TK activity and guanylate cyclase activity are currently the most clinically useful. Examples of these two receptor-linked enzymes include the following:

Receptors with integral TK activity

• Characteristic of hormones linked to metabolism, growth, and differentiation, such as insulin and epidermal growth factor

• Examples of clinical utility include:

Insulin therapy in diabetic patients

Trastuzumab (monoclonal antibody to HER2 receptor) for treatment of breast cancer

Receptors coupled to guanylate cyclase

• Generate cGMP

• Characteristic of natriuretic peptide receptors

• Example of clinical utility includes:

Agonists at these receptors are used in the treatment of heart failure. A recombinant form of brain natriuretic peptide (nesiritide) is used to reduce pulmonary congestion in acute decompensated heart failure because of the drug’s potent vasodilator properties.

Transmembrane ion channels. Transmembrane ion channels allow the passage of ions from one side of a membrane to another. Channels can exist in the open, closed, or inactive state, which represent different conformations of the channel protein. As shown in Figure 1-7, drugs may affect the function of these channels by directly opening or closing the channel (ligand gated channels), by influencing the voltage-dependent characteristics of the channels (voltage gated channels) and the amount of time the channel spends in a given state, or by generating second messengers that subsequently open or close the channel (second messenger gated). Common examples of functions governed by ion channels include the following:

Electrophysiology of cardiac and skeletal muscle

Neurotransmission

Figure 1-7 Receptor-coupled transmembrane ion channels. A, Ligand gated or receptor-operated channel. B, Voltage gated channel. C, Second messenger gated channel.

There are several subtypes of ion channels, based on the ways that drugs or endogenous substances regulate the channels (see Figure 1-7).

Ligand gated ion channels or receptor-operated channels. These ion channels possess a receptor for an endogenous ligand to which the drug can bind. They are composed of a multimeric protein complex that constitutes both the receptor and the ion channel. Agonist activation opens the channel, and antagonists close or inactivate the channel. Examples include the following:

• Cholinergic receptors located in skeletal muscle bind nicotine, resulting in opening of sodium channels, initiation of an action potential in the muscle, and finally muscle contraction. Neuromuscular (paralyzing) drugs antagonize this nicotinic receptor, thereby preventing muscle contraction.

• γ-Aminobutyric acid (GABA) A receptors are inhibitory receptors in the brain. Drugs that stimulate GABA_A receptors open chloride channels, causing hyperpolarization (making the cell more negative) and reducing the probability of an action potential being produced, thereby turning off the target neuron. Drugs that treat anxiety and sleep disorders are clinical examples of these types of drugs.

Voltage gated ion channels. These ion channels change conformation (open, closed, or inactive) in response to changes in membrane voltage. Drug binding to the channel alters the response of the channel to changes in membrane voltage such that the open, closed, or inactivate state may be lengthened or shortened. An example is a local anesthetic agent that binds to sodium channels that are responsive to the arrival of an action potential. Local anesthetics lock the channels in the inactive state, thereby rendering them temporarily nonresponsive to future action potentials and thereby block transmission of pain signals.

Second messenger gated ion channels. These ion channels respond directly or indirectly to second messenger molecules (see second messenger section, later, for details). Drug binding to receptor elaborates a second messenger that in turn affects channel function. Examples of clinical utility include the following:

• Drugs that block the hyperpolarization cyclic nucleotide gated channel (HCN or funny channel) in the sinoatrial node and thereby reduce heart rate. This class of drug represents a new approach to the treatment of angina.

Membrane-Bound Transporters

Membrane-bound transporters control movement of substances between intracellular and extracellular space and therefore control a wide range of physiologic functions. Consequently drugs that act at membrane-bound transporters have a wide range of utility including the following:

Treatment of heart failure (digitalis inhibition of sodium potassium ATPase).

Diuretics. The loop diuretics, a major class of diuretic agents, inhibit the sodium, potassium, two chloride co-transporter in the thick ascending limb of the loop of Henle, promoting loss of sodium and water in the urine.

Antidepressants. Selective serotonin inhibitors, a major class of antidepressant medication, block the reuptake of serotonin into neurons.

Gastrointestinal disorders (e.g., peptic ulcer). Proton pump inhibitors slow the secretion of hydrogen ions into the stomach and reduce gastric acidity.

Intracellular Transduction Mechanisms

A number of drugs bind to their primary site of action after being transported or diffusing into the intracellular space of the cell. Once inside the cell these receptors may be coupled to a number of transduction mechanisms, including transcriptional regulation, second messenger generation, and structural mechanisms.

Intracellular Receptors

Lipophilic drugs passively cross the cell membrane and thus do not require cell membrane receptors. As shown in Figure 1-8, one target for these drugs is an intracellular receptor that activates transcriptional pathways. In this mechanism, the agonist receptor complex diffuses to DNA, where it binds to DNA binding elements. Via this mechanism drugs act directly or through recruitment of coactivators or co-repressors, which increase or decrease transcription of RNA to ultimately change protein expression. This process is referred to as ligand gated transcriptional regulation. In many cases these drugs effect long-term changes by affecting gene transcription. Receptors using this coupling mechanism include:

Sex hormones: estrogen, androgens

Glucocorticoids

Mineralocorticoids

Thyroid or retinoid receptor family

Vitamin D receptors

Figure 1-8 Intracellular receptor-coupling mechanisms.

Responses to these types of drug may be tissue dependent based on differential recruitment of coactivators or co-repressors. For example, selective estrogen receptor modulators (SERMs) acting on the same receptor may behave as an agonist in bone and as an antagonist in breast tissue (e.g., raloxifene).

Examples of ligand gated transcription regulation with clinical utility include:

Replacement therapy (e.g., at menopause)

Contraception

Treatment of osteoporosis (via vitamin D)

Treatment of breast cancer with SERMs

Clinical Connection: Tamoxifen and raloxifene are selective estrogen receptor modulators. Tamoxifen and raloxifene act as antagonists in breast tissue and are useful in the treatment of breast cancer. However, use of tamoxifen is complicated by estrogen receptor agonist activity in the uterus, where the drug triggers an increased risk for uterine cancer. Raloxifene, despite combining to the same receptor, does not exhibit agonist activity in the uterus, and therefore its use is not complicated by concerns about uterine cancer.

Structural Mechanisms

Drugs can also target structural components of cells (e.g., the cytoskeleton or microtubules) to affect their function (see Figure 1-8). Examples of clinical utility include the following:

Vinca alkaloids (e.g., vincristine) disrupt microtubule formation and arrest cell division and are used in cancer chemotherapy.

DNA cross-linking agents (e.g., cisplatin) that inhibit DNA replication and transcription are also used in cancer chemotherapy.

In addition to activation of receptors, drugs may also target postreceptor events in second messenger cascades. For example, some drugs used in the treatment of heart failure inhibit the degradation of cAMP by blocking the PDE enzymes responsible for degradation of cAMP. Similarly, cGMP is an important second messenger molecule. Drugs used in the treatment of erectile dysfunction act by inhibiting PDE5, which is responsible for cGMP degradation. The phospholipase C (PLC), inositol trisphosphate (IP3), diacylglycerol (DAG) pathway is involved in many cellular processes. Drugs under development as antineoplastic agents target this pathway.

Cyclic Adenosine Monophosphate Pathway

cAMP is generated by activity of adenylate cyclase.

Adenylate cyclase is modulated by activated G_α-GTP complexes in either a stimulatory (G_αs-GTP) or inhibitory (G_αi-GTP) fashion.

Multiple isoforms of adenylate cyclase show isoform-specific interactions with G_α-GTP and tissue-specific distribution.

Agonists activating adenylate cyclase may elicit differential responses in different target tissues.

cAMP signaling can proceed through:

cAMP-dependent protein kinases triggering phosphorylation of effector molecules (e.g., cardiac voltage gated calcium channel) to elicit short-term effects

cAMP-dependent protein kinases triggering phosphorylation of cAMP response element binding protein (CREB), which subsequently affects gene transcription to effect long-term responses

Direct interaction of cAMP with effectors such as the hyperpolarization-activated cyclic nucleotide gated (HCN) channel that are present in cardiac pacemaker cells

PDEs play a critical role in cAMP signaling by degrading cAMP and serve to shut off signaling via this pathway. Many isoforms of PDE exist, some of which are specific for cAMP and/or show selective tissue distributions.

PDE3 inhibitors are used in the treatment of heart failure.

PDE4 inhibitors are used in the treatment of chronic obstructive pulmonary disease.

Clinical Connection: Knowledge of coupling and second messenger systems can help in understanding drug action. Clinical use of β-adrenergic antagonists is associated with two apparently disparate effects. In the short term, β antagonists reduce cardiac function by blocking the formation of cAMP and signaling to the cardiac calcium channel. However, when used chronically in heart failure patients, β antagonists actually improve cardiac function, presumably via their long-term actions on gene transcription.

Cyclic Guanosine Monophosphate

Production of cGMP through activation of guanylate cyclase, which can be:

Intrinsic to the receptor (e.g., atrial natriuretic peptide receptor)

In a soluble cytoplasmic form that serves as a target for gaseous molecules (e.g., nitric oxide)

Activation of guanylate cyclase leads to increased cGMP as a second messenger molecule that activates downstream cGMP-dependent kinases to cause phosphorylation of effector systems. An important effector system is the contractile apparatus of vascular smooth muscle, where cGMP phosphorylation leads to vasodilation.

PDEs also play a critical role as an off switch for this pathway. Accordingly, PDE inhibitors are used to interact with this signaling pathway. The development of drugs that selectively inhibit PDE5 (e.g., sildenafil), which is selective for cGMP, revolutionized the treatment of erectile dysfunction by improving penile blood flow.

Phospholipase C, Inositol 1,4,5 Trisphosphate (IP3), Diacylglycerol (DAG)

Phospholipase catabolizes membrane phospholipids to release IP3 and DAG.

IP3 serves as a second messenger controlling intracellular calcium release.

DAG activates the protein kinase C (PKC) family of enzymes.

Activation may be isoform and tissue specific.

The IP3-DAG-PKC pathway is very widespread and linked to many functions. This signaling mechanism is coupled to receptors for some of the major homeostatic pathways, including α-adrenergic receptors, serotonin receptors, angiotensin receptors, acetylcholine receptors, and prostaglandins, to name but a few. The widespread nature of this pathway makes it very important but also very difficult to manipulate to achieve selective therapeutic actions with minimal side effects. Nevertheless, this is an area of ongoing research, and isoform-specific inhibitors of PKC are in development for clinical applications. Enzastaurin, a selective PKC-β isoform inhibitor, is in clinical trials as an antineoplastic agent.

Many more second messenger systems and signaling modalities exist and participate in drug responses. Improved understanding of this facet of pharmacology will point to greater opportunities for development of new drug targets.

Clinical Connection: Knowledge of coupling and second messenger systems can help in understanding drug interactions. Nitrates used in the treatment of coronary ischemia stimulate guanylate cyclase to produce cGMP. PDE5 inhibitors used in the treatment of erectile dysfunction inhibit the breakdown of cGMP. Concurrent use of these two drugs can lead to excessive levels of cGMP, which in turn cause excessive vasodilation and potentially dangerous reductions in blood pressure. Consequently, patients are warned to not use PDE5 inhibitors if they are on nitrate therapy for angina.